Novel proteins and novel genes encoding the same

ABSTRACT

Novel proteins, novel genes encoding the same, plasmids respectively comprising these genes, transformants respectively comprising these plasmids, antibodies or fragments thereof against the above novel proteins, methods of detecting a bacterial infection, and novel polynucleotides are disclosed. The novel proteins are activated human macrophage-specific proteins.

TECHNICAL FIELD

The present invention relates to plural novel proteins, a novel geneencoding each of the proteins, a plasmid comprising each of the genes, atransformant comprising each of the plasmids, an antibody or a fragmentthereof against each of the novel proteins, a method for detecting abacterial infection, and novel polynucleotides. The novel proteins ofthe present invention are activated human macrophage-specific proteins.

BACKGROUND ART

It is known that a Lipopolysaccharide (LPS) is a glycolipid existing inan outer membrane of Gram-negative bacterium and activates a macrophageto induce an expression of many genes. Known examples of such geneshaving expressions that are induced by the LPS are those of interleukin(IL)-1, exhibiting an antitumor function or an inflammatory function,such as a function to cause an inflammation by a bacterial infection,IL-6, IL-12, IL-15, IL-18, a tumor necrosis factor (TNF), or a chemokine(such as IL-8 or MCP); granulocyte colony-stimulating factor (G-CSF)exhibiting a hemapoietic function, monocyte (M)-CSF, or GM-CSF; orcollagenase playing a main role in an inflammation such as that causedby a bacterial infection, cyclooxygenase (COX), or a nitrogen oxidesynthase (iNOS) or the like. Almost all of the above proteins encoded bythe above genes are physiologically active proteins playing importantroles in a body [Annu. Rev. lmmunol., 2, 283-318 (1984); Inflammation:Basic Principles and Clinical Correlates, 637-662, Raven Press Ltd., NewYork (1992)].

There are about 0.1 million genes in the human chromosome, but only 10or 20 percentage thereof have been isolated and identified. Therefore,as almost all of the genes have not been isolated or analyzed, it isbelieved that almost all of the genes having an expression that isspecifically induced by LPS are unidentified novel genes.

Septicemia is a systemic disease wherein a festering lesion exists in abody, and many bacteria are intermittently or continuously introducedinto the blood from the festering lesion. A diagnosis of the septicemiais carried out by culturing the blood, and when the existence ofbacteria is proved, the illness is definitely diagnosed as thesepticemia. However, the above method has disadvantages, namely, themethod is time-consuming, and when a blood sample is drawn, it may becontaminated with bacteria from skin, such as Staphylococcusepidermidis.

Therefore, the inventors of the present invention made an intensivesearch for genes having an expression that is induced specifically at amacrophage by an LPS-stimulation, for the purpose of an application fordeveloping a new method of diagnosis and/or medicament for treating adisease such as inflammation, allergy, or cancer, particularly abacterial infection. As a result, three novel genes were isolated andidentified. Further, the present inventors found that these three geneswere not expressed in healthy persons, but were expressed in patientssuffering from a bacterial infection. The invention is based on theabove findings.

DISCLOSURE OF INVENTION

The present invention relates to

-   (1) a protein comprising an amino acid sequence of SEQ ID NO: 2 in    the sequence listing, or a variation functionally equivalent    thereto, or a fragment of the protein or the variation (hereinafter    sometimes collectively referred to as a “first novel protein of the    present invention”),-   (2) a protein comprising an amino acid sequence of SEQ ID NO: 4 in    the sequence listing, or a variation functionally equivalent    thereto, or a fragment of the protein or the variation (hereinafter    sometimes collectively referred to as a “second novel protein of the    present invention”), and-   (3) a protein comprising an amino acid sequence of SEQ ID NO: 6 in    the sequence listing, or a variation functionally equivalent    thereto, or a fragment of the protein or the variation (hereinafter    sometimes collectively referred to as a “third novel protein of the    present invention”).

Further, the present invention relates to

-   (1) a gene encoding the above-mentioned “first novel protein of the    present invention” (hereinafter sometimes referred to as a “first    novel gene of the present invention”), (2) a gene encoding the    above-mentioned “second novel protein of the present invention”    (hereinafter sometimes referred to as a “second novel gene of the    present invention”), and-   (3) a gene encoding the above-mentioned “third novel protein of the    present invention” (hereinafter sometimes referred to as a “third    novel gene of the present invention”).

Further, the present invention relates to plasmids comprising each ofthe above-mentioned genes.

Further, the present invention relates to transformants comprising eachof the above-mentioned plasmids.

Further, the present invention relates to antibodies or fragmentsthereof, characterized by being reactive specifically to each of theabove-mentioned proteins or variations functionally equivalent thereto.

Further, the present invention relates to a method for detecting abacterial infection, characterized by analyzing the proteins or thevariations functionally equivalent thereto, or the mRNAs thereof, in asample to be detected.

Further, the present invention relates to

-   (1) a polynucleotide capable of specifically hybridizing to an mRNA    consisting of an base sequence of SEQ ID NO: 1 in the sequence    listing (hereinafter sometimes referred to as a “first probe of the    present invention”),-   (2) a polynucleotide capable of specifically hybridizing to an mRNA    consisting of an base sequence of SEQ ID NO: 3 in the sequence    listing (hereinafter sometimes referred to as a “second probe of the    present invention”), and-   (3) a polynucleotide capable of specifically hybridizing to an mRNA    consisting of an base sequence of SEQ ID NO: 5 in the sequence    listing (hereinafter sometimes referred to as a “third probe of the    present invention”).

The term “variation functionally equivalent” as used herein means aprotein having an amino acid sequence wherein one or more (particularlyone or several) amino acids are deleted in, changed in, or added to theamino acid sequence of an original protein, and exhibiting the sameactivities as the original protein. The term “added” as used hereinincludes an addition of one or more (particularly one or several) aminoacids to an N-terminus and/or a C-terminus of an amino acid sequence,and an insertion of one or more (particularly one or several) aminoacids to an inside of an amino acid sequence.

Further, the term “homologous protein” as used herein means a proteincomprising an amino acid sequence having a 90% or more (preferably 95%or more, more preferably 98% or more, most preferably 99% or more)homology with the amino acid sequence of an original protein, andexhibiting the same activities as the original protein. The term“homology” as used herein means a value calculated by BLAST [Basic localalignment search tool; Altschul, S. F. et al., J. Mol. Biol., 215,403-410, (1990)].

Furthermore, the terms “gene” and “polynucleotide” as used hereininclude both of DNA and RNA.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of electrophoresis wherein the expression ofthree novel genes of the present invention in human macrophagesstimulated by LPS or not stimulated by LPS was detected by a northernblotting method.

FIG. 2 shows the results of electrophoresis wherein the tissue-specificexpression of three novel genes of the present invention was detected bya northern blotting method.

FIG. 3 provides micrographs showing the results of the expression of thegene NLG-1-1 of the present invention in COS-1 cells.

FIG. 4 provides micrographs showing the results of the expression of thegene NLG-2 of the present invention in COS-1 cells.

FIG. 5 provides micrographs showing the results of a FISH analysis ofthe gene NLG-1-1 of the present invention.

FIG. 6 provides photographs showing the results of electrophoresiswherein the expression of three novel genes of the present invention inhealthy persons and patients suffering from septicemia was detected by anorthern blotting method.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

The first novel protein of the present invention includes

-   (1) a protein comprising an amino acid sequence of SEQ ID NO: 2 in    the sequence listing,-   (2) a variation functionally equivalent to the protein (1),-   (3) a protein homologous to the protein (1), and-   (4) a fragment thereof [i.e., a fragment of the protein (1), the    variation (2), or the homologous protein (3)]. A protein consisting    of the amino acid sequence of SEQ ID NO: 2 in the sequence listing,    or a variation functionally equivalent or protein homologous thereto    are preferred.

The protein consisting of the amino acid sequence of SEQ ID NO: 2 in thesequence listing consists of 481 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 2 in the sequencelisting has a high homology of approximately 82% in the amino acidsequence with mouse IRG-1 (lmmune-responsive protein-1) [Immnogenetics,41, 263-270, (1995)], and thus seems to be human IRG-1.

In the amino acid sequence of SEQ ID NO: 2 in the sequence listing,there exist eight known sites to be phospholylated by protein kinase Cand ten known sites to be phospholylated by casein kinase C. As aresult, it is believed that the protein consisting of the amino acidsequence of SEQ ID NO: 2 in the sequence listing plays an important rolein an intracellular signal transduction system which transducesinformation of LPS-stimulation.

Further, a signal peptide sequence at the N-terminus does not exist, andthus, it is believed that the protein consisting of the amino acidsequence of SEQ ID NO: 2 in the sequence listing exhibits biologicalactivities in cells.

As the protein comprising the amino acid sequence of SEQ ID NO: 2 in thesequence listing, there may be mentioned, for example, a fusion proteinof the protein consisting of the amino acid sequence of SEQ ID NO: 2 inthe sequence listing with a fusion partner. In the fusion protein, thefusion partner may be linked with the N-terminus and/or C terminus ofthe protein consisting of the amino acid sequence of SEQ ID NO: 2.

As the fusion partner, for example, a protein for purification such asthe whole or a part of glutathione-S-transferase (GST), a protein fordetection such as the whole or a part of β-galactosidase α peptide (LacZα), or a protein for expression such as a signal sequence may be used.

Further, in the fusion protein, an amino acid sequence which may berestrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 2 in the sequence listing and thefusion partner.

The fragment of the protein comprising the amino acid sequence of SEQ IDNO: 2 in the sequence listing or the variation functionally equivalentor protein homologous thereto is not particularly limited, so long as itmay be used as an immunogen to prepare the first antibody or fragmentthereof according to the present invention, but preferably consists of13 or more amino acid residues, more preferably 20 or more amino acidresidues, most preferably 50 or more amino acid residues.

The first novel protein of the present invention may be obtained byvarious known methods. For example, the protein may be prepared by usinga known genetic engineering technique and the first novel gene of thepresent invention.

The first novel gene of the present invention is not particularlylimited, so long as it encodes the first novel protein of the presentinvention. As the gene, there may be mentioned, for example, a geneconsisting of the 37th to 1479th bases in the base sequence of SEQ IDNO: 1 in the sequence listing.

The gene consisting of the 37th to 1479th bases in the base sequence ofSEQ ID NO: 1 in the sequence listing encodes the protein consisting ofthe amino acid sequence of SEQ ID NO: 2 in the sequence listing.Further, the gene consisting of the 37th to 1479th bases in the basesequence of SEQ ID NO: 1 in the sequence listing is not expressed inhealthy persons, but is expressed in patients suffering from a bacterialinfection.

The first probe of the present invention is not particularly limited, solong as it is capable of specifically hybridizing to an mRNA consistingof the base sequence of SEQ ID NO: 1 in the sequence listing. As theprobe, there may be mentioned, for example, a single or double strandedpolynucleotide consisting of a base sequence complementary to that ofSEQ ID NO: 1 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the first probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 2180 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting, under the conditions described in Example 1(4). In thoseconditions, it is twice washed with 2×SSC (standard sodium citrate)containing 0.1% sodium dodecyl sulfate (SDS) at room temperature for 20minutes, and further twice washed with 0.2×SSC containing 0.1% SDS at65° C. for 20 minutes.

The second novel protein of the present invention includes

-   (1) a protein comprising an amino acid sequence of SEQ ID NO: 4 in    the sequence listing,-   (2) a variation functionally equivalent to the protein (1),-   (3) a protein homologous to the protein (1), and-   (4) a fragment thereof [i.e., a fragment of the protein (1), the    variation (2), or the homologous protein (3)]. A protein consisting    of the amino acid sequence of SEQ ID NO: 4 in the sequence listing,    or a variation functionally equivalent or protein homologous thereto    are preferred.

The protein consisting of the amino acid sequence of SEQ ID NO: 4 in thesequence listing consists of 390 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 4 in the sequencelisting has a high homology of approximately 82% in the amino acidsequence with mouse IRG-1 (lmmune-responsive protein-1) [Immnogenetics,41, 263-270, (1995)], and thus seems to be human IRG-1.

In the amino acid sequence of SEQ ID NO: 4 in the sequence listing,there exist six known sites to be phospholylated by protein kinase C andeight known sites to be phospholylated by casein kinase C. As a result,it is believed that the protein consisting of the amino acid sequence ofSEQ ID NO: 4 in the sequence listing plays an important role in anintracellular signal transduction system which transduces information ofLPS-stimulation.

Further, a signal peptide sequence at the N-terminus does not exist, andthus, it is believed that the protein consisting of the amino acidsequence of SEQ ID NO: 4 in the sequence listing exhibits biologicalactivities in cells.

As the protein comprising the amino acid sequence of SEQ ID NO: 4 in thesequence listing, there may be mentioned, for example, a fusion proteinof the protein consisting of the amino acid sequence of SEQ ID NO: 4 inthe sequence listing with a fusion partner. In the fusion protein, thefusion partner may be linked with the N-terminus and/or C terminus ofthe protein consisting of the amino acid sequence of SEQ ID NO: 4.

As the fusion partner, for example, a protein for purification such asthe whole or a part of glutathione-S-transferase (GST), a protein fordetection such as the whole or a part of β-galactosidase α peptide (LacZα), or a protein for expression such as a signal sequence may be used.

Further, in the fusion protein, an amino acid sequence which may berestrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 4 in the sequence listing and thefusion partner.

The fragment of the protein comprising the amino acid sequence of SEQ IDNO: 4 in the sequence listing or the variation functionally equivalentor protein homologous thereto is not particularly limited, so long as itmay be used as an immunogen to prepare the second antibody or fragmentthereof according to the present invention, but preferably consists of13 or more amino acid residues, more preferably 20 or more amino acidresidues, most preferably 50 or more amino acid residues.

The second novel protein of the present invention may be obtained byvarious known methods. For example, the protein may be prepared by usinga known genetic engineering technique and the second novel gene of thepresent invention.

The second novel gene of the present invention is not particularlylimited, so long as it encodes the second novel protein of the presentinvention. As the gene, there may be mentioned, for example, a geneconsisting of the 126th to 1295th bases in the base sequence of SEQ IDNO: 3 in the sequence listing.

The gene consisting of the 126th to 1295th bases in the base sequence ofSEQ ID NO: 3 in the sequence listing encodes the protein consisting ofthe amino acid sequence of SEQ ID NO: 4 in the sequence listing.Further, the gene consisting of the 126th to 1295th bases in the basesequence of SEQ ID NO: 3 in the sequence listing is not expressed inhealthy persons, but is expressed in patients suffering from a bacterialinfection.

The second probe of the present invention is not particularly limited,so long as it is capable of specifically hybridizing to an mRNAconsisting of the base sequence of SEQ ID NO: 3 in the sequence listing.As the probe, there may be mentioned, for example, a single or doublestranded polynucleotide consisting of a base sequence complementary tothat of SEQ ID NO: 3 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the second probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 1970 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 3 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 3 in the sequencelisting, under the conditions described in Example 1(4).

The third novel protein of the present invention includes

-   a protein comprising an amino acid sequence of SEQ ID NO: 6 in the    sequence listing,-   (2) a variation functionally equivalent to the protein (1),-   (3) a protein homologous to the protein (1), and-   (4) a fragment thereof [i.e., a fragment of the protein (1), the    variation (2), or the homologous protein (3)]. A protein consisting    of the amino acid sequence of SEQ ID NO: 6 in the sequence listing,    or a variation functionally equivalent or protein homologous thereto    are preferred.

The protein consisting of the amino acid sequence of SEQ ID NO: 6 in thesequence listing consists of 83 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 6 in the sequencelisting has a homology of approximately 27% in the amino acid sequencewith mouse NADH-ubiquinoneoxidoreductase MLRQ subunit (CI-MLRQ). It isreported that the mouse NADH-ubiquinoneoxidoreductase MLRQ subunitexists in the complex I, one of four complexes I, II, III, and IV whichform an electron transport system of a mitochondrion, and takes part ina production of an active oxygen [Circulation Res., 85, 357-363 (1999);Biochem. Mol. Biol. Int., 43, 669-675 (1997)]. The protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing does nothave a gap, as suggested from the amino acid sequence thereof, and it isassumed that the protein has a structure similar to that of the mouseNADH-ubiquinoneoxidoreductase MLRQ subunit. Therefore, the protein maycontain an electron transport activity and take part in a production ofan active oxygen upon inflammation.

Further, a signal peptide sequence at the N-terminus does not exist, andthus, it is believed that the protein consisting of the amino acidsequence of SEQ ID NO: 6 in the sequence listing exhibits biologicalactivities in cells.

As the protein comprising the amino acid sequence of SEQ ID NO: 6 in thesequence listing, there may be mentioned, for example, a fusion proteinof the protein consisting of the amino acid sequence of SEQ ID NO: 6 inthe sequence listing with a fusion partner. In the fusion protein, thefusion partner may be linked with the N-terminus and/or C terminus ofthe protein consisting of the amino acid sequence of SEQ ID NO: 6.

As the fusion partner, for example, a protein for purification such asthe whole or a part of glutathione-S-transferase (GST), a protein fordetection such as the whole or a part of β-galactosidase α peptide (LacZα), or a protein for expression such as a signal sequence may be used.

Further, in the fusion protein, an amino acid sequence which may berestrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing and thefusion partner.

The fragment of the protein comprising the amino acid sequence of SEQ IDNO: 6 in the sequence listing or the variation functionally equivalentor protein homologous thereto is not particularly limited, so long as itmay be used as an immunogen to prepare the third antibody or fragmentthereof according to the present invention, but preferably consists of13 or more amino acid residues, more preferably 20 or more amino acidresidues, most preferably 50 or more amino acid residues.

The third novel protein of the present invention may be obtained byvarious known methods. For example, the protein may be prepared by usinga known genetic engineering technique and the third novel gene of thepresent invention.

The third novel gene of the present invention is not particularlylimited, so long as it encodes the third novel protein of the presentinvention. As the gene, there may be mentioned, for example, a geneconsisting of the 56th to 304th bases in the base sequence of SEQ ID NO:5 in the sequence listing.

The gene consisting of the 56th to 1304th bases in the base sequence ofSEQ ID NO: 5 in the sequence listing encodes the protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing.Further, the gene consisting of the 56th to 1304th bases in the basesequence of SEQ ID NO: 5 in the sequence listing is not expressed inhealthy persons, but is expressed in patients suffering from a bacterialinfection.

The third probe of the present invention is not particularly limited, solong as it is capable of specifically hybridizing to an mRNA consistingof the base sequence of SEQ ID NO: 5 in the sequence listing. As theprobe, there may be mentioned, for example, a single or double strandedpolynucleotide consisting of a base sequence complementary to that ofSEQ ID NO: 5 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the third probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 652 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 5 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting, under the conditions described in Example 1(4).

The plasmid of the present invention is not particularly limited, solong as it comprises the novel gene of the present invention, i.e., thefirst novel gene of the present invention, the second novel gene of thepresent invention, or the third novel gene of the present invention. Forexample, it may be a respective plasmid prepared by incorporating eachof the above genes of the present invention into a known vector suitablyselected depending on a host cell used, that is, the first plasmid ofthe present invention comprising the first novel gene of the presentinvention, the second plasmid of the present invention comprising thesecond novel gene of the present invention, and the third plasmid of thepresent invention comprising the third novel gene of the presentinvention.

The transformant of the present invention is not particularly limited,so long as it comprises the plasmid of the present invention, i.e., thefirst plasmid of the present invention, the second plasmid of thepresent invention, or the third plasmid of the present invention. Forexample, it may be a transformant prepared by transforming a desiredhost cell with each of the plasmids of the present invention, that is,the first transformant comprising the first plasmid of the presentinvention, the second transformant comprising the second plasmid of thepresent invention, or the third transformant comprising the thirdplasmid of the present invention.

The host cell may be, for example, a known microorganism usually used,for example, E. coli or Saccharomyces cerevisiae, or a known cultivatedcell, such as an animal cell, such as a CHO cell or a COS cell, or aninsect cell such as a BmN4 cell.

The known expression vector may be, for example, pUC, pTV, pGEX, pKK, orpTrcHis for E. coli; pEMBLY or pYES2 for a yeast; pMAMneo for a CHOcell; pcDNA3 for a COS cell; a vector (such as pBK283) containing apolyhedrin promoter of a silkworm nucleopolyhederovirus (BmNPV).

The first antibody of the present invention or the fragment thereof isreacted specifically with the first protein of the present invention orthe variation functionally equivalent thereto, respectively. The secondantibody of the present invention or the fragment thereof is reactedspecifically with the second protein of the present invention or thevariation functionally equivalent thereto, respectively. The thirdantibody of the present invention or the fragment thereof is reactedspecifically with the third protein of the present invention or thevariation functionally equivalent thereto, respectively.

The antibody of the present invention may be a monoclonal antibody or apolyclonal antibody.

The respective monoclonal antibodies of the present invention, that is,the first monoclonal antibody of the present invention specificallyreactive with the first protein or the variation functionally equivalentthereto of the present invention, respectively, the second monoclonalantibody of the present invention specifically reactive with the secondprotein or the variation functionally equivalent thereto of the presentinvention, respectively, or the third monoclonal antibody of the presentinvention specifically reactive with the third protein or the variationfunctionally equivalent thereto of the present invention, respectively,may be prepared by a method which is in itself known, except that thenovel protein of the present invention, the variation functionallyequivalent thereto, or a fragment thereof is used as an immunogen or anantigen for a screening.

For example, a hybridoma secreting the monoclonal antibody of thepresent invention may be prepared by immunizing a mouse with the aboveimmunogen, fusing a splenic cell taken from the immunized mouse and amouse myeloma cell in accordance with a cell-fusion method disclosed inNature, 256, 495 (1975), or an electric cell-fusion method disclosed inJ. Immunol. Method, 100, 181-189 (1987), and carrying out a screeningwith the antigen for a screening as above.

As the medium for cultivating the hybridomas, any medium suitable for acultivation of a hybridoma may be used. Preferably, the Dulbecc'smodified Eeagle's minimum essential medium containing fetal calf serum,L-glutamine, L-pyruvic acid, and antibiotics (penicillin G andstreptomycin) may be used.

The cultivation of the hybridoma may be carried out in 5% CO₂ and at370° C. for about 3 days in a medium, or for about 14 days in theabdominal cavities of mice.

It is possible to isolate or purify the monoclonal antibody from theresulting culture liquid or mouse ascites, using a method generallyapplied for the isolation and purification of proteins.

As examples thereof, there may be mentioned ammonium sulfate saltingout, ion exchange column chromatography using ion exchange cellulose,molecular sieve column chromatography using molecular sieve gel,affinity column chromatography using protein A binding polysaccharides,dialysis, lyophilization, or the like.

The respective polyclonal antibodies of the present invention, that is,the first polyclonal antibody of the present invention specificallyreactive with the first protein or the variation functionally equivalentthereto of the present invention, respectively, the second polyclonalantibody of the present invention specifically reactive with the secondprotein or the variation functionally equivalent thereto of the presentinvention, respectively, or the third polyclonal antibody of the presentinvention specifically reactive with the third protein or the variationfunctionally equivalent thereto of the present invention, respectively,may be also prepared by a method which is in itself known, except thatthe novel protein of the present invention, the variation functionallyequivalent thereto, or a fragment thereof is used as an immunogen or anantigen for a screening by, for example, the following method.

That is, a physiological salt solution containing an antigen is mixedwith an equal volume of complete Freund's adjuvant or incompleteadjuvant, or an equivalent thereof, such as Hunter's TiterMax™(Funakoshi; Cat. No. YT001-00, Tokyo, Japan), until emulsified. Theresulting emulsion is administered subcutaneously, intraperitoneally, orintramuscularly to a mammal, for example, a rabbit or goat (a firstimmunization). Then, the same procedure is repeated at intervals of twoto four weeks for several immunizations. One or two weeks after a finalimmunization, blood is taken from a carotid artery or a heart of themammal, and salted-out with ammonium sulfate to prepare a serum.

Each of the antibody-fragments of the present invention is notparticularly limited, so long as it is a partial fragment of theantibody (including the monoclonal antibody and the polyclonal antibody)of the present invention, and has a specific reactivity that is the sameas that of the original antibody. The fragment of the present inventionmay be, for example, Fab, Fab′, F(ab′)₂, or Fv. The antibody fragment ofthe present invention may be prepared, for example, by digesting thepolyclonal antibody or monoclonal antibody of the present invention witha known protease by a conventional method, and then isolating andpurifying by a conventional method.

The inventors of the present invention found that the protein of thepresent invention, particularly, the protein consisting of the aminoacid sequence of SEQ ID NO: 2 in the sequence listing, the proteinconsisting of the amino acid sequence of SEQ ID NO: 4 in the sequencelisting, or the protein consisting of the amino acid sequence of SEQ IDNO: 6 in the sequence listing, and the mRNA thereof, is not expressed ina healthy person but in a patient suffering from a bacterial infection,such as septicemia, pneumonia, urinary tract infection, myelitis, ortympanitis. Therefore, the proteins of the present invention or mRNAsthereof may be used as a diagnostic marker of a patient suffering from abacterial infection. More particularly, when an in vitro detectionmethod of the present invention finds an existence of the protein of thepresent invention and/or mRNA thereof in a sample taken from a subject,the subject can be judged to be a patient suffering from a bacterialinfection. On the contrary, when the protein and/or the mRNA is notfound, the subject can be judged to be a person not suffering from abacterial infection.

The sample which may be used in the present invention is notparticularly limited, so long as it has a possibility of including theprotein of the present invention and/or the mRNA thereof. The sample maybe a biological sample taken from an animal, such as a mammal,particularly a human (particularly a patient), for example, a tissue(e.g., cells) or an extract therefrom, blood such as serum or plasma,urine, or a humor such as cerebrospinal fluid. A sample used in aconventional clinical examination may be used in the present inventionwithout limitation.

The method of the present invention will be explained hereinafter withrespect to the method for detecting a bacterial infection by analyzingthe mRNA of the protein of the present invention, and then, with respectto the method for detecting a bacterial infection by analyzing theprotein of the present invention.

In the methods of the present invention, the method for detecting abacterial infection by analyzing the mRNA of the protein of the presentinvention is not particularly limited but, for example, may be a methodcomprising steps of bring a sample into contact with a polynucleotidecomprising a base sequence complementary to the base sequence of themRNA of the protein of the present invention; and analyzing a coupledproduct of the polynucleotide and the mRNA of the protein of the presentinvention (hereinafter referred to as a “first detecting method of thepresent invention”), or a method comprising the steps ofreverse-transcribing an mRNA in a sample to a cDNA, amplifying genes inaccordance with a gene-amplifying reaction, particularly, a polymerasechain reaction (PCR), using a reaction product obtained in thereverse-transcribing step, and primers which may amplify genes with thegene encoding the present protein as a template, and analyzing theamplified genes in the above gene-amplifying step (hereinafter referredto as a “second detecting method of the present invention”).

In the first method for detection of the present invention, the sampleis reacted with a polynucleotide (for example, the probe of the presentinvention) comprising a base sequence complementary to that of the mRNAof the protein of the present invention, and the resulting complex ofthe polynucleotide and “the mRNA of the protein of the presentinvention” is detected, or the amount of the complex is measured tothereby analyze the mRNA of the protein of the present invention.

The polynucleotide comprises a sequence complementary or substantiallycomplementary to that of a part of the mRNA transcribed from a selectedgene (DNA), and thus forms a double strand with the mRNA transcribedfrom the target gene. It is believed that any polynucleotidesufficiently complementary to form a stable complex with a target mRNAcan be used. The polynucleotide able to be used in the present inventionmay be complementary to substantially any region in a target mRNA. Thepolynucleotide can be used as a DNA probe for detecting an increase or adecrease of an expression of the mRNA specific to the gene of theprotein according to the present invention. That is, the polynucleotideis specifically attached to the mRNA of the protein according to thepresent invention as a target, and forms a molecular hybrid, whereby adegree of expression of the mRNA of the protein according to the presentinvention in cells can be detected.

The polynucleotide able to be used in the first method for detection ofthe present invention may be prepared by appropriately selecting a basesequence complementary to a specific base sequence of the mRNA of theprotein according to the present invention, and using a known DNAsynthesizer, a PCR apparatus, a gene cloning or the like. Various lengthpolynucleotides may be used, but the polynucleotide preferably has 10 ormore bases, more preferably 17 or more bases.

The polynucleotide may be a non-modified polynucleotide or apolynucleotide analogue. An appropriate analogue may be, for example, anethyl or methyl phosphate analogue, or a phosphorothioatedpolydeoxynucleotide [Nucleic Acids Res., 14, 9081-9093, (1986); J. Am.Chem. Soc., 106, 6077-6079, (1984)], with recent improvement in theproduction of polynucleotide analogue, for example, a2′-O-methylribonucleotide [Nucleic Acids Res., 15, 6131-6148, (1987)],or a conjugated RNA-DNA analogue, i.e., chimera polynucleotide [FEBSLett., 215, 327-330, (1987)], may be used.

The selected polynucleotide may be of any kind, for example, may have anelectrical charge or no electrical charge. The polynucleotide may belabeled with a known labeling agent, such as a radioactive isotope, or afluorescent substance by a conventional method, so as to carry out theabove experiment in vitro or in vivo. The radioactive isotope may be,for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³²P, or ³⁵S. Of these radioactiveisotopes, it is preferable to label the polynucleotide with ³²P by arandom primer method [Anal. Biochem., 132, 6-13, (1983)]. Further, afluorescent coloring agent forming a derivative may be used as alabeling agent, as this enables an easy handling with a low risk factor.As the fluorescent coloring agent, any coloring agents capable ofbinding the polynucleotide may be used. For example, fluorescein,rhodamin, Texas red, 4-fluoro-7-nitrobenzofurazane (NBD), coumarin,fluorescamine, succinyl fluorescein, or dansyl may be preferably used.

An amount of an MRNA of the protein according to the present inventionmay be measured by a northern blotting method, using cDNA of the proteinaccording to the present invention as follows: an mRNA is extracted andisolated from any somatic cell or tissue, then the isolated mRNA iselectrophoresed on an agarose gel and transferred onto a nitro celluloseor nylon membrane, and then reacted with a cDNA probe of the proteinaccording to the present invention to measure an amount of the mRNA ofthe protein according to the present invention. The cDNA probe of theprotein according to the present invention as used is a DNAcomplementary to the mRNA of the protein according to the presentinvention, and has preferably 17 or more bases.

In the reverse-transcribing step and the gene-amplifying step(particularly, the PCR step) of the second detecting method according tothe present invention, the reactions per se may be carried out inaccordance with the conventional reverse-transcribing method and theconventional gene-amplifying method, for example, areverse-transcription PCR (RT-PCR). More particularly, areverse-transcriptase and oligo(dT) primers are used to carry out thereverse-transcription. Then, a thermostable DNA polymerase, such as aTaq polymerase, is used to carry out an initial denaturing reaction, forexample at 97° C. for 2 to 3 minutes. Subsequently, an amplifying cycleconsisting of (1) a step for denaturing DNAs at 90 to 94° C. for 30seconds, (2) a step for annealing single-strand DNAs and primers at 50to 55° C. for 30 seconds, and (3) a step for synthesizing DNAs by thethermostable DNA polymerase at 70 to 75° C. for 1 to 2 minutes isrepeated, for example 15 to 45 times, to perform the PCR.

The analyzing step of the second detecting method according to thepresent invention can be carried out in accordance with, for example, aconventional analyzing method, for example, a method comprising steps ofcarrying out agarose-gel electrophoresis and then staining the gel witha suitable DNA-binding colorant such as ethidium bromide, or a southernblotting, or the like

The method for detecting a bacterial infection by analyzing the proteinof the present invention, one of the methods according to the presentinvention, is not particularly limited but, for example, may comprisesteps of bringing a sample into contact with an immunoreactive substancewhich can immunologically react with the protein of the presentinvention, and analyzing a coupled product of the immunoreactivesubstance and the protein of the present invention (hereinafter referredto as a “third detecting method of the present invention”).

In the third detecting method of the present invention, the sample isbrought into contact with the immunoreactive substance which mayimmunologically react with the protein of the present invention. When asample from a human is used, the sample is preferably brought intocontact with an immunoreactive substance which can immunologically reactwith the protein consisting of the amino acid sequence of SEQ ID NO: 2,the protein consisting of the amino acid sequence of SEQ ID NO: 4, orthe protein consisting of the amino acid sequence of SEQ ID NO: 6.

When the sample is brought into contact with the substanceimmunologically reactive to the protein of the present invention, if thesample does not contain the protein of the present invention, a reactionwith the immunologically reactive substance does not occur. If thesample contains the protein of the present invention, theimmunologically reactive substance binds the protein of the presentinvention, and a complex of the immunologically reactive substance andthe protein of the present invention is formed in an amount correlatedwith that of the protein of the present invention present in the sample.The complex may be easily detected by a known method, and therefore, anexistence of the protein of the present invention in the sample can bedetected by detecting the existence of the complex, or an amount of theprotein of the present invention in the sample can be measured bymeasuring the amount of the complex. The protein of the presentinvention in a tissue or a cell may be measured by using a tissuesection sample or a cell sample in a fluorescent antibody technique oran enzyme antibody technique.

The immunologically reactive substance capable of immunologicallyreacting the protein of the present invention includes an antiserumagainst the protein of the present invention, a polyclonal antibodyagainst the protein of the present invention, or a monoclonal antibodyagainst the protein of the present invention, or a fragment of theseantibodies. The immunologically reactive substance may be used singly orin a combination thereof. The fragment includes, for example, Fab, Fab′,F(ab′)₂, or Fv.

In the third method for detection according to the present invention,the sample is brought into contact with the immunologically reactivesubstance capable of immunologically reacting the protein of the presentinvention, and a complex of the protein in the present invention and theimmunologically reactive substance is formed. Then, the protein in thepresent invention bound to the antibody is detected and the amountthereof is measured by an immunochemical method, to thereby find a levelof the protein of the present invention in the sample.

Principally, the immunochemical method may be, for example, anyconventional immunoassay, for example, EIA, ELISA, RIA or the like. Theimmunochemical methods are generally classified as follows:

(1) Competitive Assay:

A sample containing an unknown amount of antigens and a given amount oflabeled antigens is competitively reacted with a given amount ofantibodies, and then an activity of the labeled antigens bound to theantibodies or an activity of the labeled antigens not bound to theantibodies is measured.

(2) Sandwich Assay:

An excess amount of antibodies immobilized on carriers is added andreacted to a sample containing an unknown amount of antigens (a firstreaction). Then, a given excess amount of labeled antibodies is addedand reacted therewith (a second reaction). An activity of the labeledantibodies on the carriers is measured. Alternatively, an activity ofthe labeled antibodies which are not on the carriers is measured. Thefirst reaction and the second reaction may be carried out at the sametime, or sequentially.

When a labeling agent is a radioactive isotope, a well counter or ascintillation counter may be used for measurement. When the labelingagent is an enzyme, an enzymatic activity can be measured by colorimetryor fluorimetry, after adding a substrate and allowing to stand. When thelabeling agent is a fluorescent substance or an luminescent substance, aknown method therefor may be used, respectively.

Recently, in addition to the above methods, a western blotting methodhas been used wherein electrophoresed proteins are transferred onto afilter such as a nitrocellulose membrane, and a target protein isdetected with an antibody. The western blotting method may also be usedin the detection of the protein according to the present invention.

The antibody used in the above methods can be labeled with anappropriate marker. Examples are a radioactive isotope, an enzyme, afluorescent substance, or a luminescent substance, by a known method oflabeling antibodies.

The radioactive isotope may be, for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C, or³⁵S.

Preferably, the enzyme used is stable and has a large specific activity.Examples of the enzyme are a glycosidase (such as, β-galactosidase,β-glucosidase, β-glucuronidase, β-fructosidase, α-galactosidase,α-glucosidase, or α-mannosidase), an amylase (such as, α-amylase,β-amylase, isoamylase, glucoamylase, or taka-amylase), a cellulase, or acarbohydrase such as lysozyme; a urease, or an amidase such asasparaginase; a choline esterase, such as acetylcholinesterase, aphosphatase, such as alkaline phosphatase, a sulfatase, an esterase suchas lipase; a nuclease such as deoxyribonuclease or ribonuclease; an ironporphyrin enzyme, such as a catalase, peroxidase or cytochrome oxidase;a copper enzyme, such as a tyrosinase or ascorbate oxidase;dehydrogenase, such as an alcohol dehydrogenase, malate dehydrogenase,lactate dehydrogenase, or isocitrate dehydrogenase.

The fluorescent substance may be, for example, fluorescamine, or afluorescence isothiocyanate, and the luminescent substance may be, forexample, luminol, a luminol derivative, luciferin or lucigenin. A signalfrom the above label may be detected by known methods.

The labeling agent can be bound to antibodies by any conventionalmethod, such as a chloramin T method [Nature, 194, 495-496, (1962)], aperiodic acid method [Journal of Histochemistry and Cytochemistry, 22,1084-1091, (1974)], or a maleimide method [Journal of Biochemistry, 79,233-236, (1976)].

An EIA method, as one of the above measurement methods will be mentionedhereinafter. A sample is added to the first antibodies immobilized on acarrier (such as an assay plate), and the first antibodies are bound tothe proteins of the present invention to form complexes. To thecomplexes, the second antibodies labeled with enzyme (such asperoxidase) are added to react with the complexes to form “firstantibody/protein of the present invention/second antibody” complexes. Tothe resulting “first antibody/protein of the present invention/secondantibody” complexes, a substrate for the enzyme label (such asperoxidase) is added, and an absorbance or fluorescent strength ofproducts of the enzymatic reaction is measured, whereby enzymaticactivities of the enzyme labels attached to the “first antibody/proteinin the present invention/second antibody” complexes are measured. Aseries of the above procedures is carried out in advance for a standardsolution containing a known amount of the protein of the presentinvention, and a standard curve based on the relationship between theprotein of the present invention and the absorbance or fluorescentstrength is prepared. A comparison is made between the standard curveand absorbance or fluorescent strength for a sample containing anunknown amount of the proteins according to the present invention, andthus, the amount of the proteins according to the present invention inthe sample can be measured.

Another EIA method will be mentioned hereinafter. A sample is broughtinto contact with a carrier (such as an assay plate) to immobilize theproteins of the present invention in the sample on the carrier. Then,the first antibodies are added thereto to form complexes of the proteinaccording to the present invention and the first antibody. To thecomplexes are added anti-first antibody antibodies (second antibodies)labeled with an enzyme (such as peroxidase), to react with the complexesto form “protein of the present invention/first antibody/secondantibody” complexes. To the resulting “protein of the presentinvention/first antibody/second antibody” complexes is added a substratefor the enzyme label (such as peroxidase), and the absorbance orfluorescent strength of products of the enzymatic reaction is measured,whereby enzymatic activities of the enzyme labels attached to the“protein of the present invention/first antibody/second antibody”complexes are measured. A series of the above procedures is carried outin advance for a standard solution containing a known amount of theprotein according to the present invention, and a standard curve basedon the relationship between the protein of the present invention and theabsorbance or fluorescent strength is prepared. A comparison is madebetween the standard curve and the absorbance or fluorescent strengthfor a sample containing an unknown amount of the proteins according tothe present invention, and the amount of the proteins according to thepresent invention in the sample is measured.

Further, an RIA method will be mentioned hereinafter. A sample is addedto the first antibodies immobilized on a carrier (such as a test tube),and the first antibodies are bound to the proteins of the presentinvention to form complexes. To the complexes are added the secondantibodies labeled with radioactive isotope (such as ¹²⁵I), to reactwith the complexes to form “first antibody/protein of the presentinvention/second antibody” complexes. A radioactivity (such asγ-radioactivity) of the resulting “first antibody/protein of the presentinvention/second antibody” complexes is measured. A series of the aboveprocedures is carried out in advance for a standard solution containinga known amount of the protein according to the present invention, and astandard curve based on the relationship between the protein of thepresent invention and the radioactivity is prepared. A comparison ismade between the standard curve and the radioactivity for a samplecontaining an unknown amount of the proteins according to the presentinvention, and the amount of the proteins according to the presentinvention in the sample is measured.

Another RIA method will be mentioned hereinafter. A sample is broughtinto contact with a carrier (such as a test tube) to immobilize theproteins of the present invention in the sample on the carrier. Then,the first antibodies are added thereto to form complexes of the proteinaccording to the present invention and the first antibody. To thecomplexes are added anti-first antibody antibodies (second antibodies)labeled with a radioactive isotope (such as ¹²⁵I), to react with thecomplexes to form “protein of the present invention/firstantibody/second antibody” complexes. A radioactivity (such asγ-radioactivity) of the resulting “protein of the presentinvention/first antibody/second antibody” complexes is measured. Aseries of the above procedures is carried out in advance for a standardsolution containing a known amount of the protein according to thepresent invention, and a standard curve based on the relationshipbetween the protein in the present invention and the radioactivity isprepared. A comparison is made between the standard curve and theradioactivity for a sample containing an unknown amount of the proteinsaccording to the present invention, and the amount of the proteinsaccording to the present invention in the sample is measured.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples.

Example 1

Isolation and Identification of Activated Human Macrophage-SpecificNovel Genes

(1) Preparation of mRNA derived from macrophages stimulated bylipopolysaccharide (LPS) From 1 liter of blood derived from a healthyperson as a starting material, peripheral blood monocytes were preparedusing a commercially available reagent for preparing peripheral bloodmonocytes (Lymphoprep; Nycomed, Oslo, Norway). The obtained peripheralblood monocytes were suspended in an RPM1640 medium containing 10 μg/mLLPS (Difco Laboratories, Detroit, Mich., USA) and 10% fetal calf serum(FCS) so that the concentration of cells became 10⁶ cells/mL. To eachplastic dish, 20 mL of the cell suspension was poured and cultured underthe condition of 37° C. and 5% CO₂.

After culturing for 3 hours, the supernatant was discarded, and adherentcells (i.e., macrophages stimulated by LPS) were washed three times with20 mL of a phosphate-buffered saline (PBS). After 3 mL of a solution forcell lysis [4 mol/L guanidine isothiocyanate, and 30 mmol/L sodiumacetate (pH 4.8)] was added, suction and ejection were repeated threetimes using a syringe with a needle. The lysate was put on 1.2 mL of a5.7 mol/L cesium chloride buffer (pH 4.8) in a ultracentrifuge 5PA tube(Hitachi Koki; Katsuta, Japan). After centrifuging for 18 hours (20° C.,38000 rpm), the supernatant in the centrifuge tube was discarded. Thepellet in the centrifuge tube was dissolved in 200 μL of sterile waterto collect RNA. From 1 L of blood, approximately 1 mg of total RNA(i.e., total RNA derived from macrophages stimulated by LPS) wasobtained.

Then, using a commercially available kit for preparing mRNA [Poly(A)Quik mRNA Isolation Kit; Stratagene, La Jolla, Calif., USA], 15 μg ofmRNA (i.e., mRNA derived from macrophages stimulated by LPS) wasprepared from 500 μg of the total RNA.

(2) Preparation of a Phage cDNA Library

A phage cDNA library was prepared using 5 μg of the obtained mRNA (15μg) derived from macrophages stimulated by LPS. Commercially availablekits (ZAP Express cDNA Synthesis Kit and ZAP Express cDNA Gigapack IIIGold Cloning Kit; Stratagene) were used to prepare the phage cDNAlibrary.

(3) Analysis of Partial Base Sequences of cDNAs

To analyze base sequences of cDNAs derived macrophages stimulated byLPS, approximately 1000 phage plaques were picked up at random and cDNAswere recovered as a phagemid by a conventional method. With respect tothe recovered approximately 1000 cDNAs derived macrophages stimulated byLPS, 400 to 500 bases from the 5′ terminus and 3′ terminus of the cDNAswere analyzed, respectively, using a commercially available kit fordetermining base sequences (Dye Terminator Cycle Sequencing kit; PerkinElmer Japan, Urayasu). The DAN homology search of the obtained sequenceswas carried out using BLAST (basic local alignment tool) in NCBI(National Center for Biotechnology Information;http://inhouse.ncbi.nlm.nih.gov) and found 63 unknown novel genes.

(4) Analysis by Northern Blotting Total RNA derived macrophagesstimulated by LPS were prepared in accordance with the procedure forpreparing total RNA derived macrophages stimulated by LPS described inExample (1). Further, total RNAs derived macrophages not stimulated byLPS were prepared in accordance with the procedure for preparing totalRNA derived macrophages stimulated by LPS described in Example (1),except for using an RPM1640 medium containing 10% FCS instead of theRPM1640 medium containing 10 μg/mL LPS and 10% FCS. The total RNAderived macrophages stimulated by LPS (10 μg/mL LPS) and the total RNAderived macrophages not stimulated by LPS were electrophoresed on aformaldehyde/agarose gel and transferred onto a nylon membrane filter bya conventional method.

The filter onto which the RNAs were transferred was heat-treated at 80°C. under reduced pressure for 2 hours; and immersed in a commerciallyavailable solution for prehybridization (Hybrisol I; Oncor,Gaithersburg, Md., USA) to perform prehybridization at 42° C. for 3hours. Then, the novel genes obtained in Example 1(3) labeled withisotope ³²P using a random primed labeling kit (Boehringer Mannheim;Germany) were respectively added, and hybridization was carried outovernight at 42° C. On the next day, the filter was twice washed with2×SSC (standard sodium citrate) containing 0.1% sodium dodecyl sulfate(SDS) at room temperature for 20 minutes, and further, twice washed with0.2×SSC containing 0.1% SDS at 65° C. for 20 minutes. The washed filterwas wrapped in a wrap, and autoradiography was performed overnight at−80° C.

As a result, it was found that, among 63 novel genes obtained in Example1(3), three genes were those whose expression was induced by theLPS-stimulation. The results of northern blotting with respect to thethree novel genes (NLG-1-1, NLG-1-2, and NLG-2) are shown in FIG. 1. InFIG. 1, the symbol “+” means “stimulated by LPS”, the symbol “−” means“not stimulated by LPS”, and “Origin” means “starting point ofelectrophoresis”. The lengths of the mRNAs of the three novel genes(NLG-1-1, NLG-1-2, and NLG-2) were approximately 2.3 kb, approximately2.3 kb, and approximately 0.7 kb, respectively.

Further, the tissue-specific expression was examined. As shown in FIG.2, the genes NLG-1-1 and NLG-1-2 were weakly expressed in all tissuesexamined [i.e., spleen (lane 1), thymus (lane 2), prostate (lane 3),testis (lane 4), ovary (lane 5), small intestine (lane 6), largeintestine (lane 7) and peripheral blood lymphocyte (lane 8)]. On thecontrary, the gene NLG-2 was strongly expressed in testis (lane 4) andlarge intestine (lane 7), but not expressed in the other tissues.

(5) Determination of Full-Length Base Sequences

The full-length base sequences of the three novel genes (LG-1-1,NLG-1-2, and NLG-2) were determined by a conventional method.

The genes NLG-1-1 and NLG-1-2 consist of 2180 bp and 1970 bp,respectively. The concrete base sequences thereof are those of SEQ IDNO: 1 and SEQ ID NO: 3 in the sequence listing, respectively. As theresult of a homology search of the genes NLG-1-1 and NLG-1-2, the 193rdto 2139th base sequence of the gene NLG-1-1 was found to be completelyidentical to the 9th to 1955th base sequence of the gene NLG-1-2. It ispresumed that two mRNAs are transcribed from a chromosomal gene inaccordance with an alternative splicing. The gene NLG-1-1 encodes aprotein consisting of 481 amino acid residues having the amino acidsequence of SEQ ID NO: 2 in the sequence listing. The gene NLG-1-2encodes a protein consisting of 390 amino acid residues having the aminoacid sequence of SEQ ID NO: 4 in the sequence listing.

Further, the gene NLG-2 consists of 652 bp, and the concrete basesequence thereof is that of SEQ ID NO: 5 in the sequence listing. Thegene NLG-2 encodes a protein consisting of 83 amino acid residues havingthe amino acid sequence of SEQ ID NO: 6 in the sequence listing.

Example 2

Expression of the Genes NLG-1-l and NLG-2 in an Animal Cell

In this example, the genes NLG-1-1 and NLG-2 were expressed using COS-1(Dainippon pharmaceutical, Suita, Osaka, Japan) as an animal cell and apQBI25-fN3rsGFP vector (Quantum biotechnologies, Montreal, Quebec,Canada) by the following procedure. This was because when the abovevector is used, a desired gene can be expressed in the form of a fusedprotein with a green fluorescent protein (GFP), and therefore, locationsof the desired gene products can be observed by tracing the greenfluorescence.

Each cDNA of genes NLG-1-1 and NLG-2 was prepared by a reversetranscription PCR (RT-PCR) method in accordance with the followingprocedure. An mRNA was prepared from human peripheral blood monocytes(PBMC) stimulated by LPS for 3 hours. A cDNA synthesized from the mRNAusing a commercially available cDNA synthesis kit (SMART PCR cDNAsynthesis kit; Clontech, Palo alto, Calif., USA) was used as a template.

As primers, an NLG-2 forward primer consisting of the sequence:5′-CACGGATCCATTCTTCGCTGAAGTCATCATGAGC-3′(SEQ ID NO: 7), an NLG-2 reverseprimer consisting of the sequence:5′-GTGGAATTCTTTGGTCACCCTTTGGACATTTTGC-3′(SEQ ID NO: 8), an NLG-1-1forward primer consisting of the sequence:5′-CACGGATCCTTCTTTACAACGAAATGATGCTCAAG-3′(SEQ ID NO: 9), and an NLG-1-1reverse primer consisting of the sequence:5′-GTGGAATTCGGAGAGATTTGTGATAGAATTATTACATGC-3′(SEQ ID NO: 10) were used.

Using a commercially available reagent for PCR (Advantage CDNApolymerase Mix; Clontech), PCR was carried out by repeating a cycleconsisting of a denaturation step (94° C., 30 seconds) and an annealingand elongation step (68° C., 2 minutes), 30 times.

The obtained PCR product was digested with restriction enzymes BamHI(Takarashuzo, Chuo-ku, Tokyo, Japan) and EcoRI (Takarashuzo), and clonedin a pQBI25-fN3rsGFP vector using a commercially available kit (DNAligation kit Ver. 2; Takarashuzo) to use for the following experiments.

On the day before a gene transfer, COS-1 cells were plated on a 6-wellplate so that the concentration of cells became 1×10⁶ cells/well. Ineach well of the 6-well plate, an autoclaved cover glass was placed, andcells were cultured on the cover glass. On the next day, the previouslyobtained vector was transferred to cos-1 cells using a commerciallyavailable reagent for transfection (LipofectAMINE reagent; Gibco BRL,Rockville, Md., USA). After 3 days from the transfer, cells on the coverglass were fixed in PBS containing 4% (v/v) formalin for 30 minutes,treated in PBS containing 0.2% (v/v) Triton X-100 for 30 minutes, andtreated in a blocking reagent (Block Ace; Dainippon pharmaceutical)containing 20% (v/v) normal goat serum (Vector Laboratories, Burlingame,Calif., USA) for 30 minutes.

For immunostaining a mitochondrion, an anti-cytochrome c antibody (SantaCruz Biotechnology, Santa Cruz, Calif., USA) and a Texas red-labeledanti-rabbit IgG antibody (Vector Laboratories) were used. Forimmunostaining an endoplasmic reticulum, an anti-calreticulin antibody(Upstate Biotechnology, Lake Placid, N.Y., USA) and a Texas red-labeledanti-rabbit IgG antibody (Vector Laboratories) were used. Forimmunostaining a Golgi apparatus, an anti-Golgi 58K protein antibody(Sigma, St. Louis, Mo., USA) and a Texas red-labeled anti-mouse IgGantibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md., USA) wereused.

For staining a nucleus, propidium iodide (Wako Pure Chemical Industries,Osaka, Osaka, Japan) was used. For staining cytoplasm, hydroethidine(Polysciences, Warrington, Pa., USA) was used.

The cover glass was mounted on a slide glass, and observed with aconfocal laser scanning microscope FV500 (Olympus Optical CompanyLimited, Chiyoda-ku, Tokyo, Japan). The state of COS-1 cells in whichthe gene NLG-1-1 was expressed is shown in FIG. 3. The state of COS-1cells in which the gene NLG-2 was expressed is shown in FIG. 4. In FIGS.3 and 4, “A” is a green fluorograph showing the expression of the fusionprotein of GFP with the protein encoded by the gene NLG-1-1 or NLG-2;“B” is a red fluorograph wherein mitochondria were stained; “C” is acombination of the above fluorographs A and B; and “D” is a figure ofdifferential calculus interference.

As shown in FIG. 3A, the expression of the gene NLG-1-1 was observedaround the nucleus. Further, the green fluorograph in FIG. 3A and thered fluorograph (the fluorograph wherein mitochondria were stained) inFIG. 3B accorded well. In FIG. 3C, the areas wherein fluorographs A andB accorded were shown in yellow. On the contrary, the fluorographwherein endoplasmic reticula were stained, the fluorograph wherein Golgiapparatus were stained, the image wherein nuclei were stained, or theimage wherein cytoplasm was stained did not accord with the greenfluorograph in FIG. 3A. Accordingly, it was found that the proteinencoded by the gene NLG-1-1 was localized in mitochondria.

Further, as apparent from FIG. 4, it was found that the protein encodedby the gene NLG-2 was localized in mitochondria, as the protein encodedby the gene NLG-1-1.

Example 3

Determination of the Chromosomal Locus of the Gene NLG-1-1

The chromosomal locus of the gene NLG-1-1 was determined by a FISH(fluorescence in situ hybridization) analysis [Chromosoma., 102, 325-332(1993)]. The gene was located on 13q22. The results were shown in FIG.5. In FIG. 5, “A” is the result of DAPI (4′, 6-diamidino-2-phenylindole)staining, and “B” is the result of the FISH signal. The arrow in FIG. 5Adenotes the chromosomal locus of the gene NLG-1-1, and the number “13”denotes that it is the thirteenth chromosome.

Example 4

Determination of Expression of the Genes NLG-1-1, NLG-1-2, and NLG-2 inpatients suffering from septicemia

From 20 mL of each blood collected from four healthy persons and fourpatients suffering from septicemia as a starting material, peripheralblood monocytes were prepared, and then approximately 20 μg of total RNAwas respectively prepared from the peripheral blood monocytes inaccordance with the procedure described in Example 1(1).

Using 10 μg of each RNA, northern blotting was carried out in accordancewith the procedure described in Example 1(4).

The results are shown in FIG. 6. In FIG. 6, (A) shows the result usingthe gene NLG-1-1 as a probe. Similarly, (B) and (C) show the resultsusing the genes NLG-1-2 and NLG-2 as a probe, respectively. Further, inFIG. 6, lanes 1 to 4 denote the results of RNAs derived from the healthypersons, and lanes 5 to 8 denote the results of RNAs derived from thepatients suffering from septicemia.

As shown in FIG. 6, three novel genes (NLG-1-1, NLG-1-2, and NLG-2) ofthe present invention were not expressed in all four healthy persons,but were strongly expressed in all four patients suffering fromsepticemia. These results show that these genes of the present inventionmay be used for the diagnosis of a bacterial infection or a judgment ofprognosis by analyzing the expression of the genes.

INDUSTRIAL APPLICABILITY

It is known that life phenomena caused by an LPS-stimulation are similarto those in inflammation. In fact, the genes having an expression thatis induced by an LPS-stimulation specifically at a macrophage playing acentral role in inflammation include almost all genes encoding proteinsleading to inflammatory diseases, for example, an inflammatory cytokine(TNF, IL-1, IL-6, IL-18), a chemokine (IL-8, MCP), a secretory protein,such as a collagenase abnormally produced at a diseased part ofrheumatoid arthritis which is an inflammatory disease, an NO synthasewhich is an intracellular protein and produces NO (nitrogen monoxide)causing an inflammation, cylooxygenese (COXII) producing aprostaglandin, an NF-kB, i.e., a gene-transcription factor participatingin an expression of a gene encoding an inflammatory protein, or thelike.

Clinical development for inhibitors of the above proteins as anantiinflammatory drug has been intensively carried out, and manyinhibitors are undergoing a clinical study. For example, an anti-TNFantibody or an inhibitor of NO synthase is already being used as amedicament for treating an inflammatory disease. Further, a clinicaldevelopment of a collagenase inhibitor as an inhibitor for a cancermetastasis, and of a COXII inhibitor as an anticancer drug is alreadybeing carried out. These suggest that there is a strong possibility thatthe novel genes having an expression that is induced by anLPS-stimulation and proteins according to the present invention alsoparticipate in the outbreak and/or deterioration of an inflammatorydisease, an allergy disease, or a cancer.

The possibility exists that the first and second novel proteins of thepresent invention take part in an intracellular signal transductionsystem of an LPS. Further, the possibility exists that the third novelprotein of the present invention takes part in an intracellular electrontransportation and/or a radical production. Therefore, the proteins asabove are different from a target of a conventional development of anantiinflammatory drug, whereas inhibitors of the proteins as above willbe a new type of antiinflammatory drug. Further, an examination of anexpression of the genes encoding the proteins as above in a humanclinical sample by means of a reverse transcription PCR (RT-PCR), anorthern blotting, a dot blotting, or a DNA microarray would make itpossible to carry out a diagnosis of an inflammation, an allergy, or acancer. Furthermore, an antibody against the protein as above could beused to carry out a diagnosis of an inflammation, an allergy, or acancer. Still further, antisense DNAs of the genes encoding the proteinsas above could be used in a treatment of an inflammation, an allergy, ora cancer (including a gene therapy).

The probe or the antibody according to the present invention may be usedto carry out a diagnosis of a bacterial infection, such as septicemia,pneumonia, urinary tract infection, myelitis, or tympanitis, or ajudgment of prognosis. The proteins of the present invention are usefulfor preparing the antibodies of the present invention, and the novelgenes, plasmids, and transformants of the present invention are usefulfor preparing the proteins of the present invention.

As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1-10. (canceled)
 11. A gene encoding a protein comprising the amino acidsequence of SEQ ID NO: 4 in the sequence listing, or a variationfunctionally equivalent thereto, or a fragment of said protein or saidvariation.
 12. The gene according to claim 11, consisting of the126^(th) to 1295^(th) bases in a base sequence of SEQ ID NO: 3 in thesequence listing.
 13. A plasmid comprising said gene according to claim11.
 14. A transformant comprising said plasmid according to claim 13.15-17. (canceled)
 18. A polynucleotide capable of specificallyhybridizing to an mRNA consisting of a base sequence of SEQ ID NO: 3 inthe sequence listing. 19-27. (canceled)
 28. A plasmid comprising saidgene according to claim 12.